High Performance Thin Layer Chromatographic (HPTLC) Analysis of Dan Shen (Salvia miltiorrhiza) Roots Grown in Different Regions of the World Bill Schoenbart, L.Ac. Abstract Context: Comparative analysis of bioactive compounds in Dan Shen (Salvia miltiorrhiza) roots grown in China and the United States Objective: Determine whether the roots of Dan Shen (Salvia miltiorrhiza) organically cultivated in the United States have a similar constituent profile to roots grown and imported from China. Research Design and Methods: Samples of Dan Shen (Salvia miltiorrhiza) were collected from a variety of Asian sources. Using High Performance Thin Layer Chromatography (HPTLC), these were then compared to samples grown organically in the United States. Results: The roots grown organically in the U.S. showed high levels of the bioactive tanshinones and salvianolic acid. In all cases, they compared favorably with the Asian samples both qualitatively and quantitatively. Both organically grown samples from the U.S. appeared to have equal or higher levels of these important compounds. Conclusions: Dan Shen (Salvia miltiorrhiza) can be successfully grown outside of China with organic agricultural methods. The harvested roots appear to have high levels of known bioactive compounds. This has positive implications for the environment, both in the reduction of pesticide and synthetic fertilizer usage, and in reducing pressure on Chinese agricultural land due to increased demand for Chinese herbs worldwide. Introduction China’s agricultural land is coming under increased pressure from development, industrialization, and the growing demand for Chinese herbs worldwide. There is also an increasing demand for locally grown organic Chinese herbs, for both environmental and quality reasons. By cultivating these herbs locally and organically, pressure can be relieved on China’s farmland and wild plant populations. When organic cultivation methods are employed, no pesticides or chemical fertilizers will be put into the local environment. It is also possible to cultivate some Chinese herbs that are endangered in the wild. This has already been done successfully in the U.S. with endangered local herbs like goldenseal. Wild populations are preserved, while the demand for the herb is met by sustainable organic cultivation.1 1 AHPA goldenseal survey measures increased agricultural production. HerbalGram. Austin, TX: American Botanical Council and the Herb Research Foundation. Spring 1999. (46) p. 66-67. 1 There is a common belief that Chinese herbs are somehow different when they are grown outside of China. In reality, the important criteria for optimum plant growth are climate, latitude, and soil conditions, not national borders. This project collected samples of processed Chinese herbs from China, Taiwan, and Hong Kong and compared them to the same processed Chinese herbs grown organically in the United States. Comparisons will be made with both qualitative and quantitative methods. Until recently, the only North American source for Chinese herbs has been suppliers who import them directly from China. For some of these herbs, there has been a significant amount of analytical chemistry conducted in laboratories in China, Japan, North America, and Europe. Since the above discussion is concerned with growing these herbs by organic methods in the United States, it is necessary to evaluate a sampling of these herbs that are grown by this means in order to compare them with the same herb grown in China. Traditionally, as well as currently, the method of evaluation has been organoleptic, performed by experienced herbalists who recognize the color, taste, smell and consistency of high quality herbs. While this remains an important means of assessing herb quality, it is still subjective. High performance liquid chromatography (HPLC) and thin layer chromatography (HPTLC) are both universally accepted methods for evaluating the chemical composition of natural products. HPLC can give a more precise quantitative image of the constituents, while HPTLC can give a more qualitative image of the chemical profile, along with a general quantitative value. By employing HPTLC to evaluate the two sets of Chinese herbs, it will be possible to make an objective comparison between them. This study looked at HPTLC analyses of the samples. A future study will more precisely quantify the results with HPLC. The herb selected for this study is Dan Shen (Salvia miltiorrhiza), a major medicinal agent in the traditional Chinese materia medica. In a 2005 study of pesticide residues in Chinese herbs purchased in the farming areas of China and the markets of Hong Kong, the Dan Shen samples contained residues of organochlorine pesticides, making it an ideal candidate for this research.2 The red-colored roots of this Chinese sage species are employed in formulas that invigorate, cool, or nourish the blood, making it an important ingredient in prescriptions for cardiovascular, gynecological, inflammatory, and psycho- emotional disorders. Modern research has also found that compounds in Dan Shen have anti-cancer effects. Traditional Energetics, Functions, and Indications for Dan Shen3 Botanical name: Salvia miltiorrhiza Pharmaceutical name: Salviae miltiorrhizae Radix 2 Leung KS, Chan K, Chan CL, Lu GH. Systematic evaluation of organochlorine pesticide residues in Chinese materia medica. Phytother Res. 2005 Jun;19(6):514-8. 3 Bensky D, Clavey S, Stoger E. Chinese Herbal Medicine – Materia Medica, 3rd Edition. 2004; pp. 602- 604. 2 Common name: Salvia root, Red Sage root Properties: Bitter, Slightly Cold Channels: Heart, Pericardium, Liver Functions: 1. Invigorates the blood and dispels stasis 2. Clears heat and soothes irritability 3. Cools the blood and reduces abscesses 4. Nourishes the blood and calms the spirit Indications: 1. A wide variety of blood stasis disorders in any part of the body. Most commonly used for problems affecting the lower abdomen, chest, or hypochondria. It is a very important herb in gynecology. 2. Especially useful for restlessness, irritability, palpitations, and insomnia due to heat entering the nutritive level. Can also be used in patterns of Heart and Kidney yin deficiency. 3. An adjunctive herb to treat sores or the early stages of breast abscess. 4. Palpitations and insomnia due to either heat entering the nutritive and blood levels or insufficient Heart blood. Dosage: 6 - 15 grams Cautions / Contraindications: Do not use in pregnancy Formulas containing Dan Shen 1. Dan Shen Yin:4 Radix Salviae Miltiorrhizae (Dan Shen): 30 grams Lignum Santali Albi (Tan Xiang): 4.5 grams Fructus seu Semen et Pericarpium Amomi (Sha Ren): 4.5 grams Actions: Invigorates the blood, dispels blood stasis, promotes the movement of qi, and alleviates pain. Indications: Abdominal or epigastric pain which may radiate upward accompanied by signs and symptoms of blood stasis and qi stagnation. 2. Qing Ying Tang:5 Cornu Bubali (Shui Niu Jiao): 30-120 grams Radix Scrophulariae Ningpoensis (Xuan Shen): 9 grams Radix Rehmanniae Glutinosae (Sheng Di Huang): 15 grams Tuber Ophiopogonis Japonici (Mai Men Dong): 9 grams Flos Lonicerae Japonicae (Jin Yin Hua): 9 grams Fructus Forsythiae Suspensae (Lian Qiao): 6 grams Rhizoma Coptidis (Huang Lian): 4.5 grams Herba Lophatheri Gracilis (Dan Zhu Ye): 3 grams Radix Salviae Miltiorrhizae (Dan Shen): 6 grams 4 Bensky D, Barolet R. Chinese Herbal Medicine – Formulas & Strategies. 1990; p.318. 5 Bensky D, Barolet R. Chinese Herbal Medicine – Formulas & Strategies. 1990; p.75. 3 Actions: Clears the nutritive level, relieves fire toxin, drains heat, and nourishes the yin. Indications: High fever that worsens at night, severe irritability and restlessness, a scarlet, dry tongue, and a thin, rapid pulse. Some patients are thirsty, some delirious, and some exhibit faint and indistinct erythema and purpura. 3. Huo Luo Xiao Ling Dan:6 Radix Angelicae Sinensis (Dang Gui): l5 grams Radix Salviae Miltiorrhizae (Dan Shen): l5 grams Gummi Olibanum (Ru Xiang): 15 grams Myrrha (Mo Yao): l5 grams Actions: Invigorates the blood, dispels blood stasis, unblocks the collaterals, and alleviates pain. Indications: Pain in various locations such as the heart, stomach, abdomen, back, leg, or arm, bruising and swelling due to traumatic injury, rheumatic pain, fixed abdominal masses, internal or external ulceration, a dark tongue or one with static points, and a wiry pulse. Bioactive Compounds in Dan Shen: Tanshinones and Salvianolic Acid The most extensively studied compounds in Dan Shen are the various tanshinones and salvianolic acid. The chromatograms in this study will show the presence and concentration of these important constituents. However, it is the whole herb and all of its compounds that have therapeutic importance. According to the American Herbal Product Association’s white paper on marker compounds, “It is rare that a single compound or group of compounds is solely responsible for the physiological effect of a botanical; rather, there are usually many types of compounds that are therapeutically relevant.”7 The presence or absence of the tanshinones and salvianolic acid are simply indicators of the potential quality and therapeutic efficacy of the samples, as well as a confirmation of their identity. There have been numerous studies on the therapeutic effect of the tanshinones and salvianolic acid. Some examples are below. Anti-cancer Effects In a study of human non-small-cell lung cancer cells, Tanshinone I significantly inhibited the migration and invasion of the cells